CA2208318A1 - Phosphotyrosine assay and peptides for use therein - Google Patents

Abstract

This invention relates to an assay for molecules capable of interacting with cytokine receptor intracellular peptides, and to peptides suitable for use in said assay. The peptides correspond to tyrosine-containing regions of the sequence of an intracellular domain of a cytokine, a hormone receptor or a JAK
Protein Tyrosine Kinase, and comprise a tyrosine which is able to be phosphorylated. In phosphorylated form, the peptides are able to bind cytoplasmic transcription factors of the STAT family.

Description

WO96/20211 PCT~S95/16988 "PHOSPHOTYROSINE ASSAY AND PEPTIDES FOR USE THEREIN"
This invention relates to an assay for molecules which are able to interact with cytokine receptor intracellular peptideg, in particular receptors of the non-protein tyrosine kinase domain type, and to peptidessuitable for use in this assay. In a particularly preferred embodiment, the assay relates to detection of molecules which are able to bind to cytokine receptors.

Backqround and Prior Art Cellular proliferation and differentiation in multicellular organisms requires precise coordination and regulation. Bin~;ng of extracellular signalling molecules, such as cytokines and growth factors, to specific cell surface receptors provides one major mechanism by which this is achieved. These receptors span the cell membrane, and are classified as to whether or not they possess an intrinsic protein tyrosine kinase domain in their intracellular region. The tyrosine kinase domain phosphorylates tyrosine residues in many intracellular proteins, providing an important step in signal transduction. Receptors which have an intrinsic protein tyrosine kinase are known as receptor tyrosine kinases.
With the advent of the Polymerase Chain Reaction (PCR) many novel members of the receptor protein tyrosine kinase family (RTR; growth factor receptors) have been identified via the highly conserved structural elements within their catalytic ~o~n~. Even though these receptors share a common cytoplasmic protein tyrosine kinase (PTR) domain, they have distinctive structural elements in their extracellular domains. These structural elements can be used to classify the RTKs into a given subfamily, and demonstrate the structural diversity that exists within the RTK family (Wilks, 1989).
The signals which are received by the transmembrane RTKs are transmitted further downstream, and eventually into the nucleus of the cell, by other signal WO 96120211 PCI~/US9S/16g88 transduction molecules which have SRC Homology 2 (SH2) domains. These SH2 domains are regions of protein sequences (modules) of approximately 100 amino acids which bind independently to phosphotyrosine residues. The b;n~;ng demonstrates high affinity and amino acid sequence specificity. The direct, sequence-specific interaction of SH2 domains with extracellular signal-induced phosphotyrosine residues has been recognised as a unifying theme in intracellular signal transduction.
The provision of high affinity and specificity in this interaction appears to be the most important factor defining the intracellular outcome of extracellular interactions. For cell-surface RTKs which are member~ of the protein tyrosine kinase (PTR) family, it is now well documented that recruitment of substrates to the activated receptor is mediated by the phosphorylation of tyrosine residues in the intracellular domain of the receptor. The specific amino acid context in which these phosphotyrosines are embedded defines which "second-wave" substrates are bound to the receptor and become substrates for its PTK
activity.
Thus these phosphotyrosine residues form a series of molecular tags to which proteins contA; n; ng SH2 domains are attracted. Association of the SH2-bearing signal transducers with the activated receptors leads in turn to the activation of the transducers, often due to tyro~ine phosphorylation by the RTR. This provides a mechanism whereby a particular receptor can select a specific subset of SH2-containing proteins to be bound, and subsequently modified and activated. Thus all signal transduction processes triggered by the activation of a RTR are coordinated by the SH2 ~n~-; n -phosphotyrosine nexus.
The JAR family of PTKs (Just Another Rinase or JAnus ~inase) were discovered by polymerase chain reaction-based screening using degenerate oligonucleotide primersbased on highly conserved PTR catalytic domain motifs. See for example Australian Patent Application No. 88229/91.

WO96120211 PCT~S95/16988 For many receptors that are not themselves PTRs, members of the JAR family of PTRs are employed as receptor "~ chAin~"~ and these molecules then act in much the same way as do the intrinsic PTR domains of the RTR family.
While JAR PTRs do not possess obvious SH2 and SH3 domains, they do have a PTR domain (JHl) and a kinase-related domain (JH2), as well as 5 other JH domains.
The cell surface receptors for a large proportion of haemopoietic growth factors (lymphok;nes), including IL-3 and IL-6, while not themselves members of the PTR family, can be grouped together with the receptors for growth hormone, prolactin, leukaemia inhibitory factor, erythropoietin and ciliary neurotrophic factor, and other hormone6 and growth factors such as oncostatin M, to form an extended family of cytokine receptors. Members of this family are characterised by the presence of four highly conserved cysteine residues in the extracellular domain, and, except for the growth hormone receptor, the characteristic sequence motif -WSRWS-, usually close to a single transm~mbrane domain. Although the cytokine receptors have little significant amino acid sequence homology within their intracellular ~- ~; n, they are clearly an evolutionarily-related set of cell surface receptors. They do not possess any protein kinase-related sequences, but it is well known that interaction of these receptors with their respective ligands triggers a ca~cade of tyrosine phosphorylation in the target cell. Thus while these receptors are not themselves members of the PTK family, recruitment of PTR activity is an important step in propagation of intracellular signalling pathways downstream of cytokine receptors, and in fact it has been shown that members of the JAK family of PTKs act as downstream transduction factors for these receptors.
These non-PTR cytokine receptors fall into two classes. Type I cytokine receptors either require ligand-induced homodimerization for signal transduction, for example erythropoietin receptor and growth hormone WO 96/20211 PCI~/US95/16988 receptor, or require recruitment of at least two distinct sub-units for effective high-affinity ligand b;n~;ng and signal transduction. In the latter group, in many cases several different ligand-specific ~ sub-units are capable of combining with the same ~ sub-unit. For example, the sub-units of the receptors for IL-3, I~-5 and GM-CSF all combine with the same ~ sub-unit.
The Type II cytokine receptors include the receptor for interferon-~ and interferon-~ (the IFN-~/~
receptor) and the interferon-~ receptor. While interferon-~ and interferon-~ appear to share a common receptor, interferon-~ binds to a separate receptor, and there is little or no sequence homology between the two groups of interferons. However, the cellular response elicited by the two receptor types shares common features, suggesting that they may recruit very similar intracellular signal transduction pathways.
Stimulation of cells by interferon molecules leads to activation of latent cytoplasmic transcription factors which migrate to the nucleus and bind specific recognition sequences located within the promoter regions of various interferon-inducible genes. In the case of IFN-~, the cytoplasmic transcription factor is designated interferon-stimulated gene factor 3~ (ISGF-3~), which in turn binds to an interferon-stimulated response element (ISRE), which is required for induction of transcription.
The ISGF-3~ complex consists of three polypeptides, formerly designated pll3, p91 and p84, but now known respectively as STAT2, STATl~ and STATl~, where STAT stands for Signal Transducers and Activators of Transcription. The STAT molecules are a family of related transcription factors. So far, 5 genes have been described in the literature, although about 10 are thought to exist.
STATl has two alternatively spliced forms, previously designated p91 or STAT91 and p84 or STAT84 respectively. They are now called STATl~ and STATl~
respectively. STAT2 is encoded by a different gene, and W096/20211 PCT~S95/16988 the protein was previously known as pll3 or STAT113.
STAT3, STAT4, STAT5 and STAT6 have been identified, and encode proteins that are also involved in an analogous manner to STATl~, STAT1~ and STAT2 in signal transcription from various cytokine receptors.
This subject has recently been reviewed by two of the present inventors (Wilks and Harpur, 1994).
As discussed in that review, a great many growth factors and cytokines utilise common intracellular signalling molecules to generate a nuclear response.
Pathways activated by EGF, PDGF, IFN-~, IFN-~, CSF-1 and IL-6 utilise homologous DNA promoter sequences and common transcription factor components. While the IFN-~ receptor utilises STAT2, STATl~ and STAT1~, the receptors for IFN-~, EGF, PDGF and CSF-1 use STAT1~ and other as yet unidentified phosphoproteins. There is also re~-~n~ncy or overlap of the DNA response elements involved. For example, a number of cytokines cause activation of DNA
b;n~;ng proteins which recognise a single region, designated the IFN-~ response region (GRR), located within the promoter of the high-affinity Fc-~ receptor gene. Thus IL-3, IL-5, IL-10 and GM-CSF all ultimately activate transcription factors which bind to sequences within a single element, further demonstrating the overlap and degeneracy involved in intracellular signal transduction.
The specific phosphotyrosine-cont~in;ng motifs on cytokine receptors which attract members of the STAT family via their SH2 domains are at present poorly defined. One exception is the STAT bi n~i ng site of the IFN-~ receptor.
The tyrosine located at amino acid position 440 in the human sequence has been shown to be the most important residue in defining the binding of STAT1~, and therefore in the commitment of the IFN-~ treated cell to respond to this cytokine in the appropriate way.
As a model, a 16-amino acid peptide was synthesized, designated Y440, whose sequence corresponds to part of the sequence of the IFN-~ receptor CA 022083l8 l997-06-l9 WO96/20211 PCT~S95/16988 (RAPTSF~Y~k~hv~VD; SEQ. ID. NO. l), a biosensor (BIAcore )-based assay using the interaction of STATl from crude cellular extracts with Y440 on a BIAcore chip was developed. The assay is simple (it can be used to assay crude cellular extracts, with Y440 on a BIAcore chip), reproducible (the signal is strong and easily read), and dependent upon the presence of phosphotyrosine in the target peptide. It has been further demonstrated that the b;n~;ng is exclusively due to STATl, and that the b;n~ing i8 both specific and of high affinity. Thus, a robust assay for agonists, antagonists, modulators and mimics of the STATl/Y440-type interaction has been devised.
Essentially the signal transduction pathway of IFN-~ has been broken down into a series of protein-protein interactions, and the most specific of them has been targeted. This assay therefore provides a means of rational drug design and/or screening for agonists, antagonists, modulators or mimics of the effects of IFN-~on cell metabolism, including but not limited to screening of natural products.
The same basic mechanism is predicted to operate for all cytokines which act through the JAR kinase-STAT
nexus. For example, the correspon~;ng STAT2 b;n~;ng site on the ~ chain of the IFN ~/~ receptor has been identified.
This i6 IFN-a R ~Y466p], correspo~;ng to residues 460-474 of the IFN-~ receptor (FLR~lNYvrr~SLRP; SEQ. ID. NO. 2).
An IFN ~/~, IL-2, IL-4, IL-5, IL-9, IL-13, IL-15, GM-CSF, growth hormone or prolactin specific drug screen is envisaged along the same lines.
Antagonists of IL-4 and IL-5 are useful as anti-atopy drugs in the treatment of conditions such as asthma and psoriasis. Antagonists of GM-CSF are useful in the treatment of inflammatory conditions such as rheumatoid arthritis. Antagonists of interferon are useful in the treatment of autoimmune diseases such as insulin-dependent diabetes mellitus. Agonists of interferon and other immune mediators are useful in the treatment of neoplastic WO 96/20211 P~ 9!ill6g88 disease, including leukaemias. Antagonists of haemopoietic growth factors are useful in the treatment of blood dysplasias involving overproduction of blood cells, e.g.
erythropoietin antagonists for treatment of overproduction of red cells, and antagonists of GM-CSF or CSF-1 for treatment of overproduction of white cells.

SummarY of the Invention According to a firgt aspect the invention provides a peptide correspon~;ng to a tyrosine-cont~;n;ng region of the sequence of an intracellular domain of a cytokine or hormone receptor or of a JAK protein tyrosine kinase, wherein said peptide comprises a tyrosine residue which is able to be phosphorylated, and in it~
phosphorylated form is able to bind a cytoplasmic transcription factor of the STAT family.
It will be clearly understood that the invention is applicable to any cytokine and its cognate STAT
molecule. For example some cytokines or hormones preferentially interact with STAT1~, some with STAT1~, and some with STAT2. The skilled person will be able to identify the most appropriate STAT molecule to use with each desired cytokine receptor phosphopeptide, using methods known in the art or described herein.
Preferably the peptide, referred to herein as the receptor peptide, is derived from a receptor for a protein selected from the group consisting of growth hormone, prolactin, leukaemia inhibitory factor, erythropoietin, ciliary neurotrophic factor, interferon-~, interferon-~, interferon-~, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, GM-CSF, CSF-1, erythropoietin, EGF, PDGF and Oncostatin M.
More preferably the peptide is derived from a receptor for a protein selected from the group consisting of growth hormone, prolactin, interferon-~, interferon-~, interferon-~, IL-2, IL-4, IL-5, GM-CSF and erythropoietin.

WO 96/20211 ~ 9!i/16988 In one particularly preferred embodiment, the invention provides a peptide having the sequence RAPTSF~Y~K~v~VD (SEQ. ID. N0. 1), correspo~;ng to residues Lys433 to Asp448 of the amino acid sequence of the interferon-~ receptor protein, which in its phosphorylated form has the ability to bind STAT1. The peptide and its phosphorylated form are referred to herein as IFN-~ R [Y440] and IFN-~ R [Y440p] receptor.
In a second particularly preferred embodiment, the peptide has the sequence FLR~lNYvrr~SLKP (SEQ. ID. N0.

2), correspon~;ng to residues Phe460 to Pro474 of the interferon-~ receptor protein, which in its phosphorylated form has the ability to bind STAT2. This peptide and its phosphorylated form are referred to herein as IFN-~ R [Y466] and IFN-~ R [Y466p] respectively.
It will be clearly understood that homologues or variants of these peptide sequences are within the scope of the invention, provided that the relevant tyrosine is still present and able to be phosphorylated, and provided that the ability of the phosphorylated peptide to bind a STAT
molecule is retained.
The peptides of the invention may be prepared by any convenient method; for example, they may be synthesised chemically, u~ing conventional solid-phase techniquec~ they may be produced by recombinant DNA method~, or they may be prepared by cleavage of the appropriate receptor protein using appropriate enzyme techniques. The receptor protein may be prepared by any convenient method.
The peptides of the invention are useful in the screening of molecules for their ability to interact with the receptor proteins. For example the peptides of the invention may be used to screen for inhibitors of interferon-~, interferon-~, interferon-~ or of other cytokines, such as interleukins, especially interleukin-4, interleukin-5 and interleukin-6, GM-CSF, G-CSF, other haemopoietins, including but not re~tricted to leukaemia inhibitory factor, and ciliary neurotrophic factor, and W096/20211 PCT~S95/16988 hormones and growth factors, such as growth hormone and EGF.
All cytokines which use the same basic signal transduction mechanisms as those of interferon-~ are suitable for use with the peptides of this invention, especially IFN-~ R [Y440p]. Thus any molecule whose activity is mediated by a STAT molecule or by the JAK-STAT
pathway is within the scope of the invention. The person skilled in the art will readily be able to test whether the receptor for a given protein belongs in this class, to identify a suitable phosphorylation site for use in the assay of the invention, and to prepare an appropriate peptide, using methods already known in the art.
The complete amino acid sequences for many cytokine or hormone receptors are already known, or can be determined using routine methods. From the sequence, tyrosine residues susceptible to phosphorylation can readily be identified. Tyrosine(s) which are important to interaction with the cognate STAT molecule can be rapidly '20 identified using routine site-directed mutagenesis. Once the relevant tyrosine residue is identified, conveniently-sized peptides can rapidly be synthesized and tested.
Accordingly, in a second aspect the invention provides a method of screening of molecules for their ability to interact with a receptor peptide, comprising the step of exposing a receptor peptide to a molecule to be screened, and measuring the degree of interaction between the peptide and the molecule.
According to a third aspect, the invention also provides a method of measuring the ability of a molecule to inhibit or promote interaction between a receptor peptide and a molecule known to be able to bind to said receptor, comprising the step of exposing the receptor peptide to a known molecule having the ability to bind to said receptor in the presence of a putative inhibitor or promoter, and measuring the ability of the putative inhibitor to inhibit or promote Raid bin~in~.

WO 96/20211 PCI~/US95/16988 For the purpo~es of this specification, the term "interact" is to be understood to encompass promoting or inhibiting the activity of the receptor peptide. Thus a molecule which interacts with a receptor peptide may act ac an agonist or antagonist of the receptor, may mimic the activity of the receptor, or may modulate its activity directly or indirectly. Moreover, the interaction may take place either during the screening process, or may take place prior to screening. Thus this aspect of the invention includes within its scope detection of interactions which take place in vitro or in vivo, before preparation of the sample which is actually subjected to the assay, as well as interactions t~k; ng place within the sample. It is particularly envisaged that cell or tissue cultures whose medium is to be assayed, or an animal from which a tissue sample or biological fluid is to be assayed, may be pretreated with the molecule to be screened for its ability to modulate the interaction between a receptor peptide with its cognate STAT.
In both the second and third aspects of the invention, the molecules to be screened may be of either synthetic, recombinant or natural origin, and may be of a wide ~ariety of structures.
In both the second and third aspects, the binding may be measured by any con~enient means. For example, either the receptor peptide or the molecule to be tested for b;n~;ng may be labelled with a detectable marker, ~uch as a radioactive label, a fluorescent label, or a marker detectable by way of an enzyme reaction. Many suitable detection systems are known in the art. Thus assay system~
which are suitable for use in the methods of the second and third aspects of the invention include, but are not limited to, immunoassays such as enzyme linked immunosorbent assay (ELISA); affinity-type assays such as those using coated microtitre plates, beads or slides, or affinity chromatography; fluorescence-acti~ated cell sorting;
biosensor assays; and bioassays.

W096/20211 PCT~S95/16988 One particularly rapid and convenient test system uses an optical biosensor, such as the BIAcore (Pharmacia Biosensor AB, Uppsala, Sweden), which enables the use of proteins or peptides immobilized to a sensor chip. The biosensor assay is very simple, reproducible and rapid, while having high specificity. The biosensor assay enables a very high throughput of samples, and is Am~n~hle to automation, and to use with relatively crude samples, such as biological fluids, or cell or tissue extracts. It 0 i8 therefore suitable for screening of natural products for their ability to inhibit or promote b;n~;ng, or for activity as agonists or antagonists of cytokines. It is also suitable for use with biological fluids, for example in clinical assays, or culture media, including medium from recombinant bacterial or cell cultures.
The BIAcore~ assay of the invention permits qualitative or quantitative analysis of the effects of putative modulators, including agonists and antagonists, on the kinetics of the STAT/STAT and STAT/receptor peptide interaction. Potentiation of STAT b;n~;ng or decrease in affinity for STAT are characteristics which may be desirable in a given potential therapeutic agent.

Detailed DescriPtion of the Invention Abbreviations used herein as are as follows:
25 BSA bovine serum albumin FCS foetal calf serum GM-CSF granulocyte-macrophage colony-stimulating factor HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid 30 HPLC high performance liquid chromatography IFN interferon IL interleukin MAb monoclonal antibody PTK protein tyrosine kinase 35 RTK receptor tyrosine kinase RU response units W096/20211 PCT~S95/16988 STAT signal transducers and activators of transcription.
The invention will now be described by way of reference only to the following non-limiting example~, and to the figures.
Figure l shows a sensorgram profile of immunological detection of the phosphorylated tyrosine-cont~in;ng peptide IFN-y R [Y440p] immobilized to a BIAcore sensor chip, using a phosphotyrosine-specific MAb 0 A l ~g/ml antiphosphotyrosine monoclonal antibody, IFN-~ R ~Y440p]-derivatised sensor chip;
0 1 ~g/ml anti-phosphotyro~ine MAb and peptide IFN-~ R [Y440]-derivatised sensor chip;
0 l ~g/ml anti-STATl monoclonal antibody, peptide IFN-~ R [Y440p]-derivatised sensor chip.
Figure 2 shows the relative response of IFN-~ R [Y440p] peptides to crude lysates of HeLa 3 cells.
Figure 3 shows the correlation between the relative response of an IFN-~ R [Y440p]-coupled biosensor chip with the presence of immunoreactive STATl in the cytosolic fraction of a HeLa S3 cell extract.
Figure 4 shows the effect of depletion of STATl from a nuclear extract of HeLa S3 cells on the response of a biosensor coupled to IFN-~ R [Y440p].
Figure 5 shows a sensorgram demonstrating immunodetection of STATl b;n~;ng to an IFN-~ R [RY440p]-coupled sensor chip.
Figure 6 shows A: Immunoblot detection of STATl in cytosolic extracts of cell lines expressing STATl, but not in a mutant cell line which doeQ
not express this protein;
B: Sensorgrams showing immunodetection of IFN-~ R [Y440p] b; n~; ng activity of these cell extracts;
C: Comparison between binding of IFN-~ R [Y440p] and IFN-~ R [Y466p] by the WO96/20211 PCT~S95/16988 cell extracts; and D: Immunoblot detection of STAT1 in peptide-affi agarose treated samples of cell extract6 by anti-STATl MAb.

5 Example 1 Immunoloqical detection of IFN-~ receptor (R) PePtides, immobilized on to activated BIAcore Sensor chiPs The 16 amino acid peptide IFN-~ R [Y440], having the sequence RAPTSFGYDKPHVLVD correspo~; ng to residues Lys433-Asp448 within the cytoplasmic ~ -; n of the IFN
receptor, and its tyrosine-phosphorylated homologue IFN-~
R [Y440p], encompassing the functionally important residue Tyr440 of the IFN-~ receptor ~ chain (Farrar et al, 1992;
Greenlund et al , 1994), were each synthesised by conventional solid-phase Fmoc chemistry, purified by either precipitation and by preparative reversed phase (RP)-HPLC, and their identity confirmed by amino acid analysis, ion spray mass spectrometry (Nash et al, 1993) and microbore RP-HPLC with on-line W spectroscopy.
The application of the BIAcore biosensor for the analysis of biomolecular interactions (Fagerstam, 1991;
Jonsson et al , 1991; Jonsson and Malmqvist, 1992), in particular for the analysis of phosphopeptide/protein interactions, has been described elsewhere (Felder et al, 1993; End et al, 1992; Panayotou et al, 1993) and in application notes issued by Pharmacia. Unless otherwise stated, the r~nn;ng buffer used in the BIAcore experiments was 20 mM HEPES, pH 7.4, 150 mM NaCl, 2.8 mM EDTA, 0.005% Tween 20. For peptide derivatisation of BIAcore sensor chips (CM5, Pharmacia), carboxyl groups of the carboxymethylated dextran matrix were activated with a 1:1 mixture of N-hyd ox~ccinimide (NHS) and 200 mM N-ethyl-N"-(3-diethylaminopropyl)-carbodiimide (EDC), both from Pharmacia, as previously described (Nice et al , 1993), and 45 ~1 of purified peptides (~98% purity by microbore RP-HPLC, ion spray mass spectrometry) at W096/20211 PCT~S95/16988 2 mg/ml in 50 mM HEPES, pH 7.5, contA;n;ng 0.15 M NaCl were injected on to the matrix at 2 ~l/min. Excess reactive NHS
groups were blocked with 30 ~l 1.0 M ethanolamine at 2.0 ~l/min. Following the immobilization the baseline response level had increased by 450 and 400 Relative Response unit~ (RU) for IFN-~ R [Y440] and IFN-~ R [Y440p], respectively, suggesting surface concentrations of 0.45 and 0.4 ng/mm2.
The phosphotyrosine-specific MAb and the anti-STAT1 MAb were diluted into BIAcore rllnn;ng buffer at 1 ~g/ml prior to injection. The anti-STAT1 MAb was provided at 0.25 mg/ml in a solution contA; n; ng 1.0 mg/ml BSA and 50% glycerol as stabilizers. The presence of these additives does not have an effect on the immunoreactivity of the MAb (see Example 5), but iB evident in the sensorgram as a substantial refractive index change.
A 35 ~l sample contA; n; ng 1 ~g/ml of a phosphotyrosine-specific MAb (Upstate Biotechnology Incorporated, Lake Placid, NY, USA. Cat. No. 05-321) was passed at 5 ~l/min over a BIAcore sensor chip which had been derivatised with approximately 0.5 ng of IFN-~receptor-derived peptide [Y440]. Following the sample pulse the chip surface was washed for 750 8 with rllnn; ng buffer followed by injection of 30 ~l of 20 mM
phenylphosphate to desorb phosphopeptide-bound MAb. The sensorgram profile is shown in Figure 1. Injection of sample, end of injection and injection of desorption solution are indicated with ~1, ~2, ~3, respectively. In a concurrent experiment an identical sample was injected over a parallel channel of the same sensor chip derivatised with the correspo~; ng tyrosine-phosphorylated peptide IFN-~ R [Y440p]. Furthermore, a non-relevant MAb (anti-STAT1 MAb, Transduction Laboratories, Lexington, RY, USA. Cat. No. G16920) was injected over the IFN-~ R [Y440p]-derivatised channel to provide a control for the BIAcore response in this and a ~ubsequent experiment (Example 5).

W096~0211 PCT~S95/16988 Example 2 BIAcore responses of immobilized IFN-~ R [Y440] to crude HeLa lysates Cell lysates were prepared as follows. Hela S3 cells were grown in RPMI +10% FCS under 5% CO2 in a 3L spinner flask (Bellco Biotechnology) to a density of 5-8x105 cells.mL~1. Cells were-stimulated for 12-16 hours (priming) or 15 minutes (acute) with 5 ng.mL~l human recombinant IFN-~ (Genentech). Cytoplasmic extracts were prepared after appropriate stimulation as described by Lock et al (1991), with the following modifications. Cells were washed in ice-cold PBS, then resuspended in ice- cold hypotonic lysis buffer [5 mM Tris.HCl pH 7.5, 2.5 mM RCl, 1 mM MgCl2, 1 mM dithiothreitol, protease inhibitors (1 mM phenylmethyl sulphonyl fluoride (PMSF) , 10 ~g/ml aprotinin, 10 ~g/ml leupeptin, 1 ~M pepstatin, 1 mM 1,10-ph~nAnthroline, 5 ~M E64) and tyrosine phosphatase inhibitor (0.1 mM sodium orthovanadate) at a density of lxlO7/ml, and then disrupted by Dounce homogenisation (to 80% disruption). Insoluble material was removed by centrifugation at 1000 g for 4 minute~. The supernatant was then diluted 2x with hypotonic lysis buffer contA;n;ng 20% sucrose, and clarified by centrifugation at 100,000 g for 30 minuteR.
Clarified supernatant was then concentrated 7 to 10-fold in an Amicon Centricon concentrator (Amicon, Cat. No. 4304). Prior to the b;n~;ng experiments, 2.5 ml aliquot~ of concentrated cell extracts were buffer eYchAnged into an assay buffer containing 50 mM HEPES, pH 7.0, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, protea~e inhibitors (10 ~M each of leupeptin and pepstatin, 5 ~M E64, 1 mM 1,10-phen~nthroline, 1 mM PMSF, 1 mM EDTA) and phosphatase inhibitors (1 mM sodium orthovanadate, 10 mM NaF) using PD10 or NAP5 desalting cartridges (Pharmacia), with approximately 95% efficacy (according to manufacturer's notes).
The freeze-dried phosphopeptide IFN-~ R [Y440p]
wa~ dissolved in a volume of 700 ~1 at 2 mg/ml into DMSO

W096t20211 PCT~S95/16988 and incubated with 500 ~1 of Affi-lO NHS-activated agarose (BioRad) for 8 hours at room temperature. Non-reacted NHS
groups of the affinity resin were blocked by incubation with 1 M ethanolamine-HCl, pH 8.5. The immobilization of peptide was monitored by microbore RP-HPLC (with on-line W -spectroscopy) of samples taken prior to and following the coupling and blocking steps, and coupling efficiency was estimated from the peak area (absorbance at 214 nm) obtained for the analysed peptide solutions. The coupling efficiency under these conditions was 20%, yielding 280 ~g peptide/ml of agarose.
For b;n~ing studies, 1.5 ml of 7-fold concentrated HeLa extract was incubated on an end-over-end rotator for 30 minutes at room temperature with 0.5 ml IFN-~ R [Y440p~-coupled agarose. Samples (100 ~1) were taken prior to the incubation and after 10, 20 and 30 minutes, and passed over an IFN-~ R [Y440p]-derivatised sensor chip at 5 ~l/minute. The BIAcore rllnn;ng buffer for these experiments was similar to the assay buffer, but did not contain protease inhibitors and glycerol.
Samples of 10-fold concentrated cytosolic extracts of HeLa cells, or dilutions thereof, were incubated either with free IFN-~ R [Y440p] phosphopeptide or with this peptide immobilized to NHS-activated agarose (Affi-gel 10, BioRad). Aliquots of the former samples and of 100,000 x g supernatants (s/n) aspirated at indicated times off the IFN-~ R [Y440p]-Affi-gel 10, or dilutions thereof, were injected at 5 ~l/minute on to BIAcore sensor chips, derivatised on parallel ~h~nnels with IFN-~ R [Y440]
or IFN-~ R [Y440p] as described in Example 1. The increase of BIAcore responses following injection of the sample plugs (Relative Response, RU) were recorded at a defined time point (after position ~2, as indicated in Example 1) and are shown in Figure 2. After each cycle, any material r: -;n;ng bound to the surface was removed by injection of a 20 ~l-pulse of 10 mM HCl containing 0.5 M NaCl.

CA 022083l8 l997-06-l9 WO 96/20211 PCT~S95/16988 Example 3 Correlation of IFN-~ R [Y440p] BIAcore response with immunoreactive STAT1 in - SE-HPLC fractions of crude cYtosolic HeLa extracts Ali~uots (0.5 ml) of 10-fold concentrates of the cytosolic fractions of HeLa cells, which had been cultured for 16 hours with 5 ng/ml IFN-~ and then stimulated with an additional 5 ng/ml IFN-~ 15 minutes prior to harvest, were fractionated at 0.25 ml/minute in assay buffer (without glycerol, protease inhibitors or NaF) on a size exclusion HPLC column which had been calibrated with various proteins of known molecular radius (Nr)~
Size exclusion-HPLC was performed at 0.25 ml/minute on a Superose-12 column (lOx300 mm, Pharmacia) using a Waters Nodel 626 Protein purification system. Protease inhibitors and NaF were omitted from the HPLC r~lnn;ng buffer, since they resulted in critically increased backpressure during the separation. Calibration of the size exclusion-HPLC column with a mixture of stAn~Ard proteins yielded a linear correlation (r=0.9) between the log Mr and retention time for the molecular weight range between 17 and 670 Kd. Coll~n fractions which were not tested immediately were stored at 4~C overnight or at -70~C for extended periods, without 1088 in b;n~;ng activity.
Analysis on the biosensor was performed in the modified assay buffer as described in Example 2. The sample rack of the instrument was kept at 14 ~ C during the course of the analysis.
Samples (35 ~1) of each eluting 1 minute fraction were passed over parallel channels of the same sensor chip derivatised with IFN-~ R [Y440p] and IFN-~ R [Y440]
peptides, respectively. The relative responses (as defined in Example 2) in each fraction are shown in Figure 3A. No response to the immobilized, non-phosphorylated IFN-~ R peptide [Y440] was seen in any of the fractions.
Remaining sample (48 ~1) in fractions 41-47, which showed WOg6/20211 PCT~S95116988 maximal BIAcore responses, was analysed by SDS-PAGE on a 12% gel, followed by immunodetection of electroblotted proteins using an anti-STATl MAb. Proteins were electrophoretically transferred to PVDF (Immobilon, Millipore Waters. Cat. No. IPVH 000 10) membrane, and then immunodetection with STATl antibody was performed under conditions specified by the manufacturer, using detection by ~nh~nced chemiluminescence (Amersham Cat. No. RPN 2109).
The results are shown in Figure 3B.
The results show that treatment of the cells with IFN-~ causeR both a net increase in IFN-~ R [Y440p] b;n~;ng (RU) and a shift of the b;n~;ng factor to an apparently higher M~, as determined by size exclusion-HPLC. This change correlates with an increased quantity and apparent Mr of immunoreactive STATl under non-denaturing conditions during size exclusion HPLC, suggesting its identity with the b;n~;ng factor.

Example 4 Decrease of IFN-~ R ~Y440P] reactivitY in STATl-depleted HeLa nuclear extracts HeLa cells were cultured as described in Example 2, but activated with IFN-~ only 15 minutes prior to extraction. Nuclear extracts were prepared as described by Dignam et al (1983), with the following modifications.
The insoluble material removed after homogenization was resuspended in High-salt Buffer [20 mM Hepe~ pH 7.9, 25% glycerol, 1.5 mM MgCl2, 1.2 M KCl, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT], and allowed to incubate for 30 minutes on ice. Nuclear matrices were removed by centrifugation for 20 minutes at 25,000 g. Nuclear extract and the anti-STATl affinity eluate were exch~nged into assay buffer, as described in Example 2. The dilution of the extract prior to affinity absorption was chosen to yield a BIAcore response within the same range a~ the other samples.
The concentrated nuclear extract of HeLa cells, ~timulated for 15 minutes with IFN-~ (IFN-~ activated WOg6nO211 PCT~S9S/16988 cell~) prior to cell ly~is and fractionation, was absorbed on to an anti-STAT1 MAb affinity resin (Transduction - Laboratorie~. Following a wA~h;ng cycle with assay buffer the affinity coll~m~ was eluted with a buffer cont~;n;ng

3 M MgCl2, 0.075 M HEPES pH 6.5, 25% ethyleneglycol. The relative BIAcore respon~es to i_mobilised IFN-y R ~Y440p]
peptide in dilutions of the extract before and after the affinity extraction, and of the col~mn eluate, were determined. The rem~;n~er of the samples used for BIAcore analysis was analysed on a 12% SDS-PAGE gel followed by immunodetection of electroblotted proteins using an anti-STAT1 MAb as described in Example 3. In addition, an aliquot of the anti-STAT1 affinity matrix after MgCl2-elution was analysed on the same gel. The results are shown in Figure 4. Panel A summarizes the BIAcore responses; Panel B ~hows the immunoblot results.

Example 5 Immunodetection of STAT1~-b;n~;n~ to an IFN-y R [Y440p] derivatised sensor chiP
The nuclear extract of IFN-~ activated HeLa cells wa~ purified on an IFN-y R ~Y440p] affinity resin and the phenylphosphate eluate of the affinity matrix was ~Ych~nged into the appropriate assay buffer, as described in Example 2. The eluate wa~ injected either directly (A, B) or after incubation with anti-STAT1 MAb (C) over a sensor chip derivatised with IFN-~ R ~Y440p]. In a second injection immediately following the fir~t sample plug and prior to the start of the dissociation phase, either buffer (A, C) or anti-STAT1 MAb (B) was injected on to the same channel of the sensor chip. Figure 5 illustrates an overlay of BIAcore responses normalised to the same baseline. The individual injections are marked with an arrow labelled "1" (first injection) and "2" (second injection). The response levels at the start of the second injection are indicated.
Activated HeLa cells were fractionated as described in Example 4, and a 4 ml sample of the nuclear WO 96/20211 PCI~/US95/16988 fraction was extracted on 0.5 ml IFN-~ R [Y440p]-agaro~e for 30 minutes at 0~C as de~cribed in Example 2. The affinity resin was washed quickly with 8 column volumes of as~ay buffer and eluted with one column volume of phenylphosphate (20 mM), collected in a single 0.5 ml fraction. The breakthrough from this column was loaded again under identical conditions. This second b; n~; ng step increased recovery from the lysate to 90%. Both eluates were pooled, and a 90 ~1 aliquot incubated for 30 minutes at room temperature with 10 ~1 of anti-STATl MAb (200 ~g/ml) diluted into as~ay buffer, or with the same volume of assay buffer. Aliquots (35 ~1) of these sample~
were passed over a IFN-~ R [Y440p]-derivatised sensor chip at 5 ~l/minute and immediately after each sample plug had left the flow cell were followed by a second 35 ~1 injection of either rllnn;ng buffer or anti-STATl MAb at 1 ~g/ml.
Thus either preincubation of cellular extracts with anti-STATl MAb prior to BIAcore analysis or injection of MAb on to IFN-~ R [Y440p] sensor chip-bound factor resulted in an increased BIAcore response. In contrast, direct injection of the MAb on to the IFN-~ R [Y440p]-derivatised sensor chip yielded no signal (Example 1), thus indicating that the primary antibody-enhanced b; n~; ng response is due to STAT1.

Exam~le 6 Absence of INF-~ R [Y440] Peptide B; n~; n~
Component in Crude Cell Extracts, Detected by BIAcore and Immunotechnoloqy Correlates with the Absence of STAT1 Protein Parental (2fTGH) and mutant (U2A, U3A, U4A) human fibroblasts [as described elsewhere (Pellegrini et al, 1989: McKendry e t al, 1991)] were grown in Dulbecco'~
Modified Eagle' 8 Medium (DMEM) supplemented with 10%
(vol./vol.) heat-inactivated foetal calf serum and 5 ~M
L-glutamine. The cells were grown to 80% confluency, and harvested, following 2 washes in ice cold PBS supplemented CA 022083l8 l997-06-l9 W096/20211 PCT~S95/16988 with 0.1 mM Na3V04, in 1 ml trypsin-versene. Following trypsinisation, cells were washed twice in DMEM plu8 10%
foetal calf serum, and twice in ice-cold PBS supplemented with 0.1 mM Na3V04. HeLa S3 cells were grown as described in Example 2. Cytoplasmic extracts were then prepared as described in Example 2.
The presence of STAT1 in samples (48 ~1) of 10-fold concentrated cytosolic extracts, prepared as described above, was analysed by SDS-PAGE on a 7.5% gel followed by immuno-detection of electroblotted proteins using an anti-STAT1 Mab (as described in Example 3). This i~ presented in Figure 6A, and shows that STAT1 is not present in line U3A.
BIAcoreTM detection of IFN-~ R [Y440p] b; n~; ng activity in the above samples was carried out as follow~.
Samples of 10-fold concentrated cytosolic extracts, prepared as described above, were incubated either with IFN-~ R [Y440p] affi agarose or IFN-~ R [Y440] affi agarose, aspirated after 30 minute~ at room temperature, and then injected at 5 ~l/minute~ onto BIAcoreTM sensor chips, derivatised on parallel channels with IFN-~ R [Y440]
or IFN-~ R [Y440p] as described in Example 1.
BIAcoreTM detection of IFN-~ R [Y440p] b; n~; ng activity in samples of 10-fold concentrated cytosolic extracts, prepared as de6cribed above, was carried out on sensor chips, derivatised on parallel channels with IFN-~ R [Y440] or IFN-~ R [Y440p] as described in Example 1. The response sensorgrams of samples aspirated after 30 minutes incubation at room temperature with IFN-~ R [Y440p] affi agarose, subtracted (BIAevaluation software ver.2.1) from the response in samples incubated with IFN-~ R [Y440] affi agarose are indicated in Figure 6B. No response to any of the extracts was detected on the IFN-~ R [Y440] derivatised channel.
Identical experiments were carried out substituting the IFN-~ R [Y440] peptide with a phosphopeptide (IFN-~ R [Y466p]) correspo~;ng to residues WO 96/20211 PCrlUS9S/16988 460-474 of the IFN-~ R (FLRClN~v~SL~P; SEQ. ID. NO. 2) and synthesised as described in Example 1. The relative BIAcore responses following injection of each cell extract were determined after 575 seconds (indicated by the arrow in Figure 6B). Figure 6C compares the responses of each cell extract to both phosphopeptides, plotted in relative response units.
Parallel aliquots of the peptide affi-agarose treated samples analysed in Figure 6B were examined by SDS-PAGE on a 7.5% gel followed by immunodetection of electroblotted proteins using an anti-STAT1 MAb as described in Example 3. The results, shown in Figure 6D, confirm that the approach taken above is valid, in that treatment of these extracts with phosphopeptide affi-agarose effectively depletes STATl protein.
Furthermore, the absence of STAT1 in U3A extracts correlates with the lack of BIAcore response depicted in the sensorgram of Figure 6B.

ExamDle 7 Other Potential STAT B; n~; n~ Sites Tyro6ine-cont~;n;ng amino acid sequences in which the tyrosine can be phosphorylated are present in a number of proteins which have been reported in the literature.
Some of these phosphotyrosine residues are located upon cytokine receptors, whereas others are found towards the C-2 5 terminal end of the STATs themselves. The putative STAT
b;n~;ng sites are summarized in Table 1, with the location of the phosphotyrosine identified in brackets. This location uses sequence numbering as indicated by mutation analysis.

WO 96/20211 r~ l6988 ::Ln Ln Ln Ln _ILn ~ ~ ~ Ln Ln ~a 0 ~ h ~ h 0 P~
h ,~ 0 ~ h ,~

~ n ~D ~ Ln ~~

m n n n n n n n n al _I
.~, ~ ~ ~ Ln t~ Ln o ~ ~
~ D O ~'CO ~1 ~ O
a) ~7 Ln ~ c~
U ~ ~ ~ ~--~ Ln ~ --~~ ~O ~ ~ O
~ Ln ~ ~
": V ~ -- ~~ ~ ~
r~ O~ O ~-- O E-l O ~ O 1~1 0 0 0 V. O
n; ~ Z,e Z U. V 4 a~ ~ ~ v p H ,~3 H ~ H ~ H H P~ H . H ~:1 H Y H
a Y ~ ~ a Y ~ -~a ~Ja~a ~a ~a ~:a. a ~a ~a ,3 rn ~i3 V~ W ~ ~ a ~ ~ ~
YVI ~ V~ V V~ V u~ :C V~ avn ~ V~ :~ Vl ~ Vl -- ~--V-- V-- ~ --~-- ~-- Y --I r~ h ~d u~
X
j~
o ~r ~ I vn o ~ ~ P4 H I _~
li3 H P4 tJ~ ~ V n U U ~
~~ U

,, o~ o o ~ ~
o~ o u p n~ ~ ~ X
U
~ U
~.~ ~~J ~,1 , ~,1 o ~, ~n r.~ ~4 _14 _I V c4 ~1 ~0 .
m -~, ~d ~
E~ C
~J
~ o ~ o~
O _ _ ~ a~ . . . _ . _ .
~o Or~ o 4 O O O
Z -I ~ Z ~ Z;
rJ~ 4 _1 1_ ~ ~ ~ ~ ~ 01 ~ E- -a H H r H 4 H ~ H L H
> O
01 P4 0l ~ Ol rn ~ rn ~ rn > rn V rn -- rn-- ~ ¢~ a--1 ~, E~ ., rn ~ ~
P_ ~ ~ X ~ ~ o -- -- ~ ~ 0 U 4 ~1 o rn n rn n n ~4 P: H
li3 ~4 ~

CA 022083l8 l997-06-l9 WO 96/20211 P~ 9~i/16g88 Only the STAT1~ Y701 i8 demonstrably a STAT
b;n~;ng site, while the other sites have been shown to be ~ strong candidates for STAT b;nA;ng by virtue of the fact that either phenylalanine substitution of the tyrosine results in 1088 of STAT bin~;ng (for example in the case of the EPO-R Y343 or the PRL-R Y580), or the sites lie in a region which appears to be required for signal transduction down-stream of a particular cytokine receptor (for example in the case of the LIF-R and gpl30). A further class of predicted sites has been derived on the basis of what is known about STAT b;n~;ng site~, coupled with what iB known in the field of cytokine receptor-mediated signal transduction (usually denoted as Harpur, 1995).

The u~e of specific components of the IFN-~signalling cascade and synthetic analogues thereof in an BIAcore biosensor-based assay to study initial steps of this pathway in detail has been described herein. As a m;n;m~l, functionally critical portion of the ~-chain of the cytokine receptor, a peptide sequence surro~n~;ng a tyrosine residue (Tyr440) which in its phosphorylated form is essential for biological responsiveness (Farrar et al, 1991, Greenlund et al, 1994) was immobilized onto the sensor chip of the biosensor. The efficacy of the coupling reaction and the integrity and accessibility of the phosphorylated tyrosine was demonstrated by monitoring, in real time, the binding of an anti-phosphotyrosine MAb to the peptide (Example 1). HeLa cell extracts and fibroblast extracts (2fTGH, U2A and U4A) were shown to contain a component that bound specifically to the sensor chip-immobilized, phosphorylated form of the receptor peptide.
Specificity was demonstrated by:
i) competition with free IFN-~ R [Y440p]
peptide ii) lack of response to IFN-~ R [Y440] peptide iii) depletion of the IFN-~ R [Y440p] b;n~;ng WO 96/20211 PCI~/US95/16988 factor from the crude extract with immobilized receptor peptide (Example 2).
iv) $ailure of extracts of U3A mutant cells, which lack STAT1, to generate a b;n~;ng response to IFN-~ ~Y440p] peptide (Example 6).

Treatment of the cells with IFN-~ causes both a net increase in IFN-~ R [Y440p] b;n~;ng (RU) and a shift of the b;n~;ng factor to an apparently higher Mr as determined by size exclusion-HPLC. This change correlates with an increased quantity and apparent Mr ~f immunoreactive STAT1, suggesting its identity with the bi nA; ng factor (Example 3).
Three approaches were taken to verify this identity. First, immunodepletion of cellular extract with an anti-STAT1 affinity resin resulted in a titratable net reduction in IFN-y R [Y440p] b; n~; ng (RU). The successful retention of STAT1 on the affinity resin was confirmed by western analysis (Example 4; Figure 4 lane 4), which also revealed "bleeding" of MAb together with antigen from the affinity resin (lanes 2, 5-7). Elution of the affinity col-~mn with 3M MgCl2 yielded a small but titrable BIAcore response, suggesting unusually high avidity of the anti-STAT1 MAb.
Second, either preincubation of cellular extracts with anti-STAT1 MAb prior to BIAcore analysis or injection of MAb on to IFN-~ R [Y440p] sensor chip-bound factor resulted in an increased BIAcore response. In contrast, injection of the MAb on to the IFN-~ R [Y440p]-derivatised sensor chip gave no signal.

Finally, application of cellular extracts from the mutant fibroblast cell line U3A, which completely lacks STAT1~ or STAT1~ proteins, failed to generate a b;n~;ng response to the IFN-~ R [Y440p] peptide. Thus it is clear that the presence of STAT1 is required for the b; n~; ng W096/20211 PCT~S95116988 response detected towards the IFN-~ R [Y440p] peptide.
Taken together, these f;n~;ngs demonstrate that - the local sequence of relatively short peptide~ contains sufficient information to enable their use as physiologically relevant affinity reagent~. Thus a wide range of biomolecular interactions can be analysed using the approach presented herein.
It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understAn~; ng, various modifications and alteration6 to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

References cited herein are listed on the following pages, and are incorporated herein by thi~
reference.

WO96r20211 PCT~S95/16988 REFERENCES

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Fagerstam, L.
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Felder, S., Zhou, M., Hu, P., Urena, J., Ullrich, A., Chaudhuri, M., White, M., Shoelson, S.E., and Schlessinger, J.
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Mol. Cell Biol., 193 13 1449-1455.

W096/20211 PCT~S9S/16988 Gouilleux, F., Wakao, H., Mimdr, M. and Groner, B.
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EMBO, 1994 13 4361-4369 Greenlund, A.C., Farrar, M.A., Viviano, B.L., and Schreiber, R.D.
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Harpur, A.G.
PhD Thesis, University of Melbourne, 1995 Hou, J., Sc~;n~ler, U., Henzel, W.J., Ho. T.C., Brasseur, M. and McKnight, S.L.
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Science, 1994 265 1710-1706 Jons~on, U., Fagerstam, L., Ivarsson, B., Johnsson, B., Karlsson, R., Lun &, R., Lofas, S., Persson, B., Roos, H., Ronnberg, I., and et al "Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology"
Biotechniques, 1991 11 620-627.

Jonsson, U. and Malmqvist, M. (1992).
Advances in Biosen~or, Vol.2. (T.on~nn: JAI Press Ltd.).

Lebrum, I.J., Ali. S., Goffin. V., Ullrich, A. and Relly, P.A.
"A single phosphotyrosine residue of the prolactin receptor is responsible for activation of gene transcription"
Proc. Natl. Acad. Sci. USA, 1995 92 4031-4035 W096/20211 PCT~S95/16988 Lock, P., Ralph, S., Stanley, E., Boulet, I., Ramsay, R., and Dunn, A.R.
"Two isoform6 of murine hck, generated by utilization of alternative translational initiation codons, exhibit different patterns of subcellular localization"
Mol. Cell Biol., 1991 11 4363-4370.

McK~n~ry, R., John, J., Flavell, D., Muller, M., Kerr, I.M., and Stark, G.R.
"High-frequency mutagenesis of human cells and characterization of a mutant unresponsive to both alpha and gamma interferons"
Proc. Natl. Acad. Sci. U.S.A., 1991 88 11455-11459.

Nash, K., Feldmuller, M., de Jersey, J., Alewood, P., and Hamilton, S. (1993).
"Continuous and discontinuous assays for phosphotyrosyl protein phosphatase activity using phosphotyrosyl peptide substrates"
Anal. Biochem., 1993 213 303-309.

Nice, E., Lackm~nn, M., Smyth, F., Fabri, L., and Burgess, A.W.
"Synergies between micropreparative high-performance liquid chromatography and an instrumental optical biosensor"
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Panayotou, G., Gish, G., End, P., Truong, 0., Gout, I., Dhand, R., Fry, M.J., Hiles, I., Pawson, T., and Waterfield, M.D.
"Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach"
Mol. Cell Biol., 1993 13 3567-3576.

WO96/20211 PCT~S9S/16988 Pellegrini, S., John, J., Shearer, M., Rerr, I.M., and Stark, G.R.
"Use of a selectable marker regulated by alpha interferon to obtain mutations in the signaling pathway"
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Shuai, R. Stark, G.R., Rerr, I.M. and Darnell, J.E.
"A single phogphortyrosine residue of STAT91 is required for gene activation of Interferon-~ n Science, 1993 261 1744-1746 Stahl, N., Parruggella, T.J., Boulton, T.G., Zhong, Z.
Darnell, J.E.J and Yancopoulos, G.D.
"Choice of STATs and other substrates specified by modular tyrosine-based motifs in cytokine receptors"
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"Stat3 and Stat4 members of the family of signal transducers and activators of transcription"
Proc. Natl. Acad. Sci. USA, 1994 91 4806-4810 WO 96120211 PCI~/US9Stl6988 SEO~N~ LISTING

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(A) LENGTH: ll (B) TYPE: amino acid (C) STRANDEDNESS: 8 ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOLnhllCAL: no (v) F~ M~NT TYPE: internal (xi) SEQ~:N~ DESCRIPTION: SEQ ID NO: 15 Ala Val A~p Gly Tyr Val Lys Pro Gln Ile LYB

(2) INFORMATION FOR SEQ ID NO. 16:
(i) SEQ~N~ C~CTERISTICS:
(A) LENGTH: ll (B) TYPE: amino acid (C) STRANDEDNESS: 8 ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOLnh~-lCAL: no (v) F~r-M~NT TYPE: internal (xi) SEQ~N~ DESCRIPTION: SEQ ID NO: 16 Asp Gly Arg Gly Tyr Val Pro Ala Thr Ile Lys

Claims (22)

1. A receptor peptide having the amino acid sequence as set forth in SEQ. ID.
NO.: 1, which corresponds to residue Lys433 to Asp448 of the amino acid sequence of the interferon-.gamma., wherein said peptide comprises a tyrosine kinase residue that upon phosphorylation binds to a cytoplasmic transcriptional factor of the STAT family.

2. A receptor peptide having the amino acid sequence as set forth in SEQ. ID.
NO.: 2, which corresponds to residue Phe460 to Pro474 of the amino acid sequence of the interferon-.gamma., wherein said peptide comprises a tyrosine kinase reside that upon phosphorylation binds to a cytoplasmic transcriptional factor of the STAT family.

3. The receptor peptide according to claim 1, wherein said peptide in its phosphorylated form binds STAT.alpha..

4. The receptor peptide according to claim 2, wherein said peptide in its phosphorylated form binds STAT2.

The receptor peptide according to claim 1, wherein said peptide is derived from a protein receptor selected from the group consisting of a growth hormone. prolactin, leukemia inhibitory factor, erythropoietin, ciliary neurotrophic factor, interferon-.gamma., interferon-.alpha., interferon-.beta., IL-2. IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, Il-13, IL-14, IL-15, GM-CSF, CSF-1, EGF, PDGF and Oncostatin M.

6. The receptor peptide according to claim 2, wherein said peptide is derived from a protein receptor selected from the group consisting of a growth hormone, prolactin, leukemia inhibitory factor, erythropoietin, ciliary neurotrophic factor, interferon-.gamma., interferon-.alpha., interferon-.beta., IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-9. IL-10, IL-11, IL-12, Il-13, IL-14, IL-15, GM-CSF, CSF-1, EGP, PDGF and Oncostatin M.

7. The receptor peptide according to claim 5. wherein said peptide is derived from a receptor protein selected rom the group consisting of a growth hormone, prolactin, interferon-.gamma., interferon-.alpha., interferon-.beta., IL-2, IL4, IL-5, GM-CSF and erythropoietin.

8 The receptor peptide according to claim 6, wherein said peptide is derived from a receptor protein selected rom the group consisting of a growth hormone, prolactin. interferon-.gamma., interferon-.alpha., interferon-.beta., IL-2, IL4, IL-5, GM-CSF and erythropoietin.

9. A method of screening a molecule for its ability to interact with the receptor peptide of claim 1, comprising exposing said receptor peptide to a molecule to be screened, and measuring the degree of interaction between said receptor peptide and said molecule.

A method of screening a molecule for its ability to interact with the receptor peptide of claim 2, comprising exposing said receptor peptide to a molecule to be screened, and measuring the degree of interaction between said receptor peptide and said molecule.

11. The method according to claim 9, wherein said peptide is derived from a protein receptor selected from the group consisting of a growth hormone, prolactin, leukemia inhibitory factor, erythropoietin, ciliary neurotrophic factor, interferon-.gamma., interferon-.alpha., interferon-.beta., IL-2, IL-3, IL-4, IL-5, IL-6.
IL-7, IL-9, IL-10, IL-11, IL-12, Il-13, IL-14, IL-15, GM-CSF, CSF-1, EGF, PDGF and Oncostatin M.

12 The method according to claim 10, wherein said peptide is derived from a protein receptor selected from the group consisting of a growth hormone, prolactin, leukemia inhibitory factor, eryhothropeotein, ciliary neurotrophic factor, interferon-.gamma., interferon-.alpha., interferon-.beta., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, Il-13, IL-14, IL-15, GM-CSF, CSF-1, EGF, PDGF and Oncostatain M.

13 The method according to receptors peptide according to claim 11, wherein said peptide is derived from a receptor protein selected rom the group consisting of a growth hormone, prolactin, interferon-.gamma., interferon-.alpha., interferon-.beta., IL-2, IL4, IL-5, GM-CSF and erythropoietin.

14 The method according to claim 12, wherein said peptide is derived from a receptor protein selected from the group consisting of a growth hormone, prolactin, interferon-.gamma., interferon-.alpha., interferon-.beta., IL-2, IL4, IL-5, GM-CSF
and erythropoietin.

15 A method of measuring the ability of a molecule to inhibit or promote interaction between the receptor peptide according to claim 1, and a molecule known to bind to said receptor peptide, comprising exposing said receptor peptide to known molecule having the ability to bind to said receptor in the presence of a putative inhibitor or promoter, and measuring the ability of the putative inhibitor to inhibit said binding.

16. A method of measuring the ability of a molecule to inhibit or promote interaction between the receptor peptide according to claim 2 and a molecule known to bind to said receptor peptide, comprising exposing said receptor peptide to a known molecule having the ability to bind to said receptor in the presence of a putative inhibitor or promoter, and measuring the ability of the putative inhibitor to inhibit said binding

17 The method of claim 15, wherein said receptor peptide is selected from the group consisting of growth hormone, prolactin, leukemia inhibitory factor, erythropoietin, ciliary neurotrophic factor, interferon-.gamma., interferon-.alpha., interferon-.beta., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL 10, IL-11, IL-12, IL 13, IL-14, IL-15, GM-CSF, CSF-1, erythropoietin, EGF, PDGF and Oncostatin M.

18 The method of claim 16, wherein said receptor peptide is selected from the group consisting of growth hormone, prolactin leukemia inhibitory factor, erythropoietin, ciliary neurotrophic factor, interferon-.gamma., interferon-.alpha., interferon-.beta., IL-2, IL-3, IL4, IL-5, IL6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, GM-CSF, CSF-1, erythropoietin, EGF, PDGF and Oncostatin M.

19. The method of claim 17, wherein said receptor is a receptor for a protein selected from the group consisting of growth hormone, prolactin, interferon-.gamma., interferon-.alpha., interferon-.beta., IL-4, IL-5, GM-CSF and erythropoietin.

20. The method of claim 18, wherein said receptor is a receptor for a protein selected from the group consisting of growth hormone, prolactin, interferon-.gamma., interferon-.alpha., interferon-.beta., IL-4, IL-5, GM-CSF and erythropoietin.

21. The method of claim 9, wherein binding to the receptor peptide is measured by a method selected from the group consisting of radioactivity detection, fluorescence detection, immunoassay, affinity assay, fluorescence-activated cell sorting, bioassay, and biosensor assay.

22. The method of claim 10, wherein binding to the receptor peptide is measured by a method selected from the group consisting of radioactivity detection, fluorescence detection, immunoassay, affinity assay, fluorescence-activated cell sorting, bioassay, and biosensor assay.